Streptomycetes are known to produce a large variety of secondary metabolites which can be applied in the manufacturing of pharmaceuticals. Examples of these pharmaceuticals belong to the classes produced by the Streptomycetes and are for instance polyketides, macrolides, anthracyclines, tetracyclins, lipopeptides and β-lactams, see further Strohl (1997).
One particular example is the production of the β-lactamase inhibitor clavulanic acid, a β-lactam compound which is produced by various microbial strains belonging to the genus of Streptomycetes such as S. clavuligerus ATCC 27064, S. jumonjinensis (GB patent 1563103), S. katsurahamanus IFO 13716 FERM 3944 (JP patent 83009679B) and Streptomyces sp. P6621 FERM 2804 (JP patent application 55162993A).
Secondary metabolism is regulated in various ways in Streptomycetes. Such regulations may comprise the carbon catabolite, ammonium or phosphate repression or any other kind of metabolite that represses the synthesis of the secondary metabolite of interest. Carbon catabolites and ammonium repress the production of cephalosporins in S. clavuligerus (Aharonowitz and Demain 1978 and 1979), nitrogen catabolites regulate the production of spiramycin in S. ambofaciens (Untrau 1994), and phosphate, ammonium and glutamate repress the production of clavulanic acid in S. clavuligerus (Romero et al. 1984). The oxygen concentration influences the regulation of ammonium and phosphate on the antibiotic synthesis by S. clavuligerus (Fang & Demain, 1995). Various types of regulations involved in cephamycin production with various Streptomycetes are described in Omstead et al (1985). High ammonium concentrations were found to repress streptonigrin biosynthesis in S. flocculus (Wallace et al. 1990). A similar negative influence of ammonia on the cefalosporin production by Streptomyces clavuligerus was already described by Aharonowitz and Demain (1979). Furthermore, as S. clavuligerus is urease positive, which urease is repressed by NH4Cl (see page 2478 of Bascaran et al (1989)) and ureum has been produced during the clavulanic acid production (Elson, 1993), the production of clavulanic acid by S. clavuligerus is described to be especially high when the concentration of ammonium is kept low (WO 96/18743).
However, otherwise than described in the patent application WO 96/18743, surprisingly a large increase in the production of clavulanic acid with a Streptomycete was found when maintaining the ammonium concentration in an optimal rather high concentration range of about minimal 50 mg/l. When this ammonium concentration range was applied, the production of clavulanic acid was improved by more than 20%.
The application of an ammonium concentration of at least 50 mg/l to obtain a surprisingly high yield of clavulanic acid has never been described or suggested in the literature.